Process for the purification of cis-16, 17-dihydroxy steroids



ited rates Patent 3,005,839 PROCESS F GE Tim PURIFICATION 0F (ITS-16,17-D1HYDROXY STEROIDS Lewis .ioseph Leeson, Park Ridge, Siegfried Arthur Muller, Cioster, and George Madison Sieger, East Paterson,

N.J., assignors to American Cyanamid Company, New

York, N.Y a corporation of Maine No Drawing. Filed Jan. 21, 1959, Ser. No. 7 88,037 12 Claims. ((31. 260397.45)

This invention relates to a process of purifying steroids.

More particularly, it relates to a method of purifying l6a,17u-dihydroxy steroids of the pregnane series esters and intermediates thereof. I Recently steroids of the pregnane series having l6a,l7udihydroxy groups present have been shown to have high glucocorticoid activity with natriuresis. The product triamcinolone described in US. Patent No. 2,789,118 is a steroid of this type. A process for the preparation of l6u,17ot-dihydroXy steroids of the pregnane series free frompther closely related steroids is desirable.

It is an object of this invention to effect the selective separation of cis-16,l7-dihydroxy steroids from mixtures containing these dihydroxy groups and other steroids as well as further impurities.

It is a further object of this invention to selectively isolate cis-l6,l7-dihydroxy steroids from fermentation media, crude filter cakes, and so forth, where related steroids might be advantageously separated or isolated.

A still further object of this invention is to effect a purification or upgrading of a particular crude cis-16,17-dihydroxy steroid.

Previously the standard operating method to separate, for example, 9a-fluoro-l1B,l6a,l7a,2l-tetrahydroxy 4 pregnene 3,20 dione from, for example, 90 fluoroll B,17a,21-trihydroxy-4-pregnene-3,20-dione involved the use of an organic solvent such as methylisobutylketone. After concentration of the extract by heating under reduced pressure, the resulting product often contained a considerable amount of the non-cis, 16a,l7a-dihydroxy steroid and necessitated the used of complicated separation and recrystallization procedures in order to obtain a pure compound. Such procedures as the concentrating of the methylisobutylketone, extract and the separating and recrystallizing of the impure product were time consuming and expensive and invariably resulted in lower product yields.

We have now found that the present invention can be carried out starting with an aqueous solution containing a mixture of steroids which is treated with boric acid and an alkali metal carbonate. The amount of boric acid and carbonate depends largely upon the quantity of the cis-16,l7-dihydroxy steroid contained in the solution. A1- ternatively, other metals or amine carbonates or hydroxides or amines can be used in place of sodium carbonate. Also alkali metal or amine borates can be used directly. The reaction is preferably carried out at a pH of about 9.0. The mixture is agitated and the insoluble material (unreacted steroids and impurities) is filtered oil. The clear filtrate is acidified, producing the regenerated free cis-l6,l7-dihydroxy steroid as an insoluble precipitate. The separation of the mixtures of steroids containing cis-l6,l7-dihydroxy groups can be accomplished by chromatographic separation as shown hereinafter in the examples.

The process of the present invention can also be carried out in the following manner: An organic solvent such as methylisobutylketone can be used to extract the desired steroids from the harvest mash. A borate ester salt can be formed at this stage or after concentration of the methylisobutylketone. In either case, the addition of an aqueous solution of borate salt results in the forma- 3,005,83h Patented Oct. 24, 1961.

tion of a cycloborate ester of those steroids containing adjacent hydroxyl groups. Such borate esters are soluble in the aqueous borate solution and all other steroids are substantially insoluble in this solution and may be removed by simple filtration. After acidification to below pH 2, subsequent transesterification with methanol permits the formation of methyl borate which forms a minimum boilingazeotrope with methanol and is easily removed by distillation, leaving the desired cis-dihydroxy steroid in pure form.

Among the suitable starting steroids utilizable in the process of the present invention are those containing adjacent cis-hydroxyl groups in the l6a,17u-positions, any mixture of steroids or impure steroid crystals containing compounds having adjacent cis-hydroxyl groups in the l6u,l7oz-positions, and steroids capable of being hydrolyzed to compounds containing adjacent cis-hydroxy groups in the 160:- and Hat-positions, these latter including steroid acetates capable of being deacetylated to form compounds having adjacent cis-hydroxyl groups in the 16a-17a-positions, steroid monoacetates capable of being deacetylated and hydrolyzed to compounds containing adjacent cis-hydroxyl groups in the l6a,l7oz-p0SitiOnS, steroid acetonides capable of being altered to form compounds containing adjacent cis-hydroxyl groups in the 16a,17a-p'ositions.

The new process of the present invention also affords a process of preparing 2l-monoesters wherein a reactionmixture containing ll-monoester and l6ct,21-diester is treated with sodium borate solution. The 2l-monoester' selectively reacts to form the l6ot,l7a-sodium borate, dissolving in the sodium borate solution, whereas the 16a,21- diester does not react with the sodium borate, and remains undissolved. Filtration then enables separation of the undissolved 16a,21-diester from the 1606,170t-SQdlllIl'1 borate, from which the 1606,170L-dlhYdIOXY steroid can be regenerated by acidification. The process also permits the preparation of mixed esters wherein after regeneration of the 16a,l7ot-dihydroxy steroid, the l6a-hydroxy group is then esterfied by an acid or anhydride producing an ester difierent from that in the 2l-position as shown hereinafter in the examples.

EXAMPLE 1 To a small glass vessel is added 0.3963 gram of 904-- fluoro-l6u-hydroxy prednisolone, 0.0667 gram of boric acid and 5 ml. of water. To this suspension is added 0.55 of 10% sodium carbonate solution. The suspension is allowed to stand for three days with occasional shaking. At the end of thisv time, solution is almost complete, and the preparation filtered clear. Dilute hydrochloric acid is added dropwise until the pH of the solution is below two. A precipitate (B) forms and is filteredo'tf, washed and dried under reduced pressure at 45. When compared to the starting material (A), the results shown as chemical assay are obtained. The sam ple is found to have a loss on drying of 2.88%. This brings the ultraviolet assay up to 100%, and the blue tetrazoliurn. value to The sample is also found to Sample (B) weighs 0.3440 gram which is equal to 0.3341 gram when corrected for loss on drying. is a yield of approximately 85%.

EXAMPLE 2 A sample of 0.487 gram of the sodium borate salt of 9oc-fiUOIO-16or-hYdTOXY-hYdlOCOIfiSOHe is dissolved in 25 ml. of water. Dilute hydrochloric acid is added until the pH is below two. The precipitate which forms is filtered 01f, washed and dried. The infrared analysis indicates the material to be 9a-flll0i'0-lGa-hYdIOXY-hYdIOCOItlSOIIe while the blue tetrazolium analysis shows the material to be 96.0% pure.

EXAMPLE 3 EXAMPLE 4 The following mixture of steroids is prepared:

G. G. G. G. Qa-fluoro-16a-hydroxyprednisolone 0.7168 0.0562 0.1477 ga-fluoro-lfia-hydroxyhydrocortisone. 5143 Hydrocortisone 0. 4633 0.2328 Prednisone 0.0695 Prednisolone 0. 0812 To both (A) and (B) is added 5 ml. of 3% boric acid solution and 3.8 ml. of 9% sodium carbonate.

To both (C) and (D) is added 1 ml. of 3% boric acid solution and 0.76 ml. of 9% sodium carbonate solution.

The mixtures are allowed to stand 48 hours with occasional At the end of this time, the undissolved material of each mixture is filtered off, washed and dried and weighed. The filtrates are each mixed with dilute hydrochloric acid until the pH is approximately two. The precipitates which result are filtered olf, Washed, dried and weighed. The results obtained from the weights and analyses of materials are shown on the following table.

mixed with 84 g. of diatomaceous earth. Each column is developed in excess of five holdback volumes. In each case, steroid (based upon a blue tetrazolium spot test) comes ofi at the solvent front. Tailing is extensive. The first portion, approximately holdback volume, is discarded due to: presence of the colored impurity. The blue tetrazoliurm positive fractions are combined, allowed to evaporate, and the well defined crystalline 9(x-fll10l0- l6a-hydroxy-hydrocortisone or 9or-fluoro-l6or-hydroxyprednisolone products are obtained. These steroids are regenerated from the ammonium and sodium borate derivatives of each. All products are insoluble in water in contrast to the borate salt starting material.

EXAMPLE 6 Using a l6ahydroxylating strain of Streptomyces r0seochromogenus such as ATCC No. 3347 as a fermentative microorganism, a fermentation medium consisting of Soybean meal 20 g., glucose 35 g., calcium carbonate 2.5 g., soybean oil 25 ml., and water to make 1000 ml, a substrate consisting of about 250 to 400 mcg. ml. fermentation concentration of 9a-fluoro-1lfi,l7a,2l-trihydroxy- 4-pregnene 3,20 dione (F2), 9rx-flll0TOhYd1OCOI'tiSOHC (FF) and a conversion time of to 90 hours; there is produced a harvest mash containing 9oc-fluOI'O-11B,16a, 170:,2l-tetrahydroxy-4-pregnene-3,20-dione (F-3) l6a-hydroxy 90: fiuorohydrocortisone (FF 16) The harvest mash is extracted with methylisobutylketone (MIBK) and assayed 239 meg/ml. of F-3 (Blue Tetrazoliurn method) and 307 meg/ml. (Polarographic method). Five serial extractions of 2.0 liters of the MIBK extract of the 9mfiuoro llfi,l6or,l7u,2l tetrahydroxy-4-pregnene-3,20-dione harvest mash are performed using 20 ml. aliquots of a 0.10 M (4% w./v.) aqueous solution of sodium tetraborate (Na B O -l0H O). These aliquots are combined and filtered. The pH of the filtered solution is adjusted to 1.5-2.0 with 2 m1. of concentrated hydrochloric acid. After standing at room temperature overnight, the solution is filtered and the crystals thus obtained are rinsed and dried under reduced pressure. The yield is 464 mg. of 90a fluoro-l1e,16m,17,21-tetrahydroxy-4-pregnene-3,20 dione cycloborate. Transesterification of 400 mg. of this impure cycloborate is accomplished by adding 25 ml. aliquots of methanol to it five different times, evaporating the 25 ml. of solution almost to dryness each time before adding the next 25 ml. aliquot of methanol. The methyl borate formed by the transesterification process is thus formed and removed, leaving 373 mg. of 9a-fluoro-llfi, 16a,17a,21 tetrahydroxy-4-pregnene-3,ZO-dione (theoretical recovery 376 mg.) Thematerial assays 982 mcg./mg.

These results indicate an excellent method of separating cis diolsfrom noncis diols with a steroid nucleus.

EXAMPLE 5 A series of columns is set up for partition chromatography using diatomaceous earth as the support and a system of ethyl acetate/water. In each column 22 ml. of equilibrated aqueous phase is used to dissolve the sample. The charge, a steroid borate, is admixed with 28 g. of diatomaceous earth and packed on top of the development portion which is 67 ml. aqueous phase ad- (BT-Spectrophotometric) and 1012 meg/mg. (Polarographic).

EXAMPLE 7 i A 10:90 crystal mixture of 9er-fluoro-11}9,17a,21-trihydroxy-4-pregnene-3,20-dione and 9ot-fll10rO11/3,16oc,,17u, 2l-tetrahydroxy-4-pregnene-3,20-dione is slurried in an 0.10 M (4% w./v.) aqueous solution of sodium tetraborate at room temperature at the pH which is normal for borate (9.0-9.2) for /z-hour. At the termination of that period, the slurry is filtered to remove insoluble materials EXAMPLE 8 Using Bacterium cyclooxidans ATCC No. 12,673, the fermentation medium Grams Glucose 20.0 Peptone 5.0 Tryptone .0 Yeast extract 5 .0 CaCO 2.5

Distilled water q.s. 1000 ml.

and a 56 mg. substrate of 9a-fluoro-11fi,l6a,l7a,21-tetrahydroxy-4-pregnene-3,20-dione and a 24 hour conversion at a pH of about 6.0, there is obtained 9u-fluoro 1 15,16u, 17a,2ltetrahydroxy 1,4 pregnadiene-3,20-dione harvest mash. A 275 ml. portion of the above harvest mash is extracted three times with 140 ml. of methylisobutylketone each time. The pooled 420 ml. of methylisobutylketone is then back-extracted five times with 4 ml. of 0.10 M (4% W./v.) aqueous solution of Na B O -10H O each time. The total ml. of sodium borate back-extract solution is adjusted to pH=1.6 with 0.4 ml. of concentrated hydrochloric acid. No precipitation or crystallization occurs. Next, a total of 5 ml. of saturated ammonium sulfate (NH SO solution is also added in 5 steps of 1 ml. each to the acidified back-extract solution, followed by a single addition of 5 ml. of saturated sodium chloride solution. After /z-hour in a chill room some crystallization is visible at the interface of the small amount of methylisobutylketone phase present.

The total 35 ml. of liquid is then extracted three times with 17 ml. of methylisobutylketone being employed each time. This extract is evaporated in a hood draft to yield 24 mg. of a product which has the same R value as 90:- fiuoro-l1,3,16a,17a,21-tetrahydroxy-l,4 pregnadiene 3, 20-dione on a paper chromatogram.

EXAMPLE 9 One gram of triamcinolone is added to 10 ml. of 0.12 N aqueous sodium tetraborate solution. This suspension is allowed to stand 24 to 48 hours with mixing. At the end of this time it is filtered free of a small amount of remaining insoluble material and the clear filtrate lyophilized. The borate salt is obtained snow white and crystalline.

EXAMPLE 10 One gram of 9u-fluoro-115,16a,17a,21-tetrahydroxy-4- pregnene-3,20-dione is added to 10 ml. of 0.12 N aqueous sodium tetraborate solution. This suspension is allowed to stand 24 to 48 hours with mixing. At the end of this time, it is filtered free of the small amount of remaining insoluble material and clear filtrate lyophilized. The borate salt is a white crystalline solid.

EXAMPLE 11 One gram of 115,16a,17 x,21-tetrahydroxy-4-pregnene-3, ZO-dione is added to 10 ml. of 0.12 N aqueous sodium tetraborate solution. This suspension is allowed to stand 24 to 48 hours with mixing. At the end of this time it is filtered free of the small amount of remaining insoluble material and clear filtrate lyophilized. The borate salt is a white crystalline solid.

EXAMPLE 12 One gram of triamcinolone is added to 4 ml. of pyridine and 0.25 ml. of acetic anhydride is introduced. The solution is allowed to stand for 4 to 5 hours at room temperature and poured into 40 ml. of cold dilute sulfuric acid. The precipitate which forms is removed, Washed, dried and added to 25 ml. of 0.1 N sodium tetraborate solution. After standing overnight with occasional mixing the insoluble material is filtered off, and dilute hydrochloric acid added to the filtrate until the pH is below two. The observed precipitate is filtered, washed with boiling Water and dried. Infrared analysisof the product obtained by the above procedure indicated it to be triamcinolone 2l-monoacetate.

EXAMPLE 13 Following the procedure of Example 12 and substituting for triamcinolone the steroid 9a-fluoro-l6u-hydroxyhydrocortisone, a pure 2l-acetoxy-9a-fiuoro-11,8,16a,17utrihydroxy-4-pregnene-3,20-dione is obtained.

EXAMPLE 14 One gram of 9u-fiuoro-l6a-hydroxyhydrocortisone is dissolved in 6 ml. of pyridine and 0.4 ml. of butyric anhydride added. The mixture is allowed to stand overnight at room temperature. The solution is poured into 25 ml. of cold dilute sulfuric acid and the observed precipitate removed and washed. It is added to 25 ml. of 0.1 N sodium tetraborate solution and permitted to stand overnight with occasional shaking. The insoluble material is filtered and to the filtrate is added dilute hydrochloric acid until the pH is below two. The observed precipitate is filtered, washed With boiling water and dried. Infrared analysis of the product obtained by the above procedure indicated it to be pure 21-butyroxy-9a-fluoro-11,8,l6ot,l7m-trihydroxy -4- pregnene 3,20- dione.

EXAMPLE 15 One gram of 9a-fiuoro-l6a-hydroxyhydrocortisone is dissolved in 8 ml. of pyridine and 0.55 grams of benzoic anhydride added. The solution was allowed to stand overnight and then poured into 35 ml. of cold dilute sulfuric acid. The precipitate is filtered, washed and added to 25 ml. of 0.1 N sodium tetraborate solution. The mixture is allowed to stand overnight, the insoluble material removed and the filtrate acidified with HCl until the pH is below two. The observed precipitate is filtered, Washed with boiling water and dried. Infrared analysis of the product obtained employing the above procedure indicated it to be pure 2l-benzoxy-9a-fluoro-11B,16a,17u-trihydroxy-4-pregnene-3,20-dione.

EXAMPLE 16 Two hundred mg. of triamcinolone 21-monoacetate is dissolved in 2 /2 ml. of pyridine and 0.2 ml. of n-butyric anhydride added. Solution is efiected by a small amount of heat. The mixture is poured into 25 ml. of dilute hydrochloric acid and the observed precipitate filtered, washed and dried. Recrystallization from chloroformpetroleum-ether produces a pure product, 16-butyroxy- 21-acetoxy-9 a-fiuoro-l l fl,l7a-dihydroxy-1,4- pregnadiene- 3,20-dione.

We claim:

1. A process for the separation and purification of 9afluoro-l6a-hydroxyprednisolone which comprises adding said compound to a solution containing boric acid and an alkali metal carbonate, filtering ofi any solids, acidifying to a pH below about two, removing the precipitate and recovering the purified product therefrom.

2. In a process for the separation and purification of 9a-fiuoro-16a-hydroxyprednisolone the step which comprises acidifying with a mineral acid an alkali metal borate salt of 9m-fluoro-16ot-hydroxyprednisolone to a pH less than two, separating the precipitate formed and recovering the purified product therefrom.

3. A process for the separation and purification of 9afluoro-l6a-hydroxyprednisolone which comprises extracting the harvest mash resulting from the fermentation of 9a-fluoro-1113,170,21-trihydroxy-4-pregnene 3,20 dione and Streptomyces roseochromogenus with methylisobutylketone, adding sodium tetraborate to form the corresponding 16,17-cycloborate ester filtering to remove any insoluble precipitate, acidifying to pH below 2. treating said ester with methanol, azeotropically distilling off the methyl borate formed and recovering said 9a-fluoro-16ahydroxyprednisolone therefrom.

4. A process for the separation and purification of 9afluoro-l6oc-hydroxyprednisolone which comprises dissolving said compound in an aqueous solution of sodium tetraborate, filtering to remove any insoluble precipitate, acidifying to a pH below 2 adding methanol, azeotropical: 1y distilling ofi methyl borate and recovering said 9afluoro-16a-hydroxy-prednisolone therefrom.

5. A process for the separation and purification of 9afluoro-l6a-hydroXy-prednisolone-2l-acetate which comprises reacting 9a-fluoro-16a-hydroXy-prednisolone with acetic anhydride precipitating the reaction product by the addition of a mineral acid, dissolving the said precipitate in aqueous alkali metal tetraborate solution, filtering off impurities, adding a mineral acid to a pH of less than two and recovering the said product in pure form.

6. A process for the separation and purification of 90:- fiuoro-16a-butyroxy2l-acetoxy-prednisolone which comprises reacting 9a-fiuoro-16oc-hydroXy-prednisolone with an equivalent of an acetylating agent, precipitating the reaction product by the addition of a mineral acid, dissolving the said precipitate in aqueous alkali metal tetraborate solution, filtering off impurities, precipitating the reaction product by the addition of a mineral acid to a pH less than two, reacting the product with butyric anhydride and recovering said product therefrom.

7. A process for the purification of 11B,l6a,17u,21- tetrahydroxy 4 pregnene 3,20 dione which comprises treating said 4-pregnene with an aqueous alkali metal borate solution to form a steroid alkali metal borate ester, separating the latter borate ester in Solution, acidifying said solution to a pH less than 2. and recovering said purified compound.

8. A process for the purification of an llfl-hydroxycis-l6a,l7a-dihydroxy A' -3,20-diketo steroid of the pregnane series which comprises mixing said steroid with an aqueous alkaline borate solution to form the steroid borate salt, separating the said borate salt in solution, acidifying the said borate salt solution to a pH less than 2 and recovering the said cis l6oc,l7a-dihydroxy steroid therefrom.

9. A process for the purification of an 11 fl-hydroxy cisl6a,l7u-dihydroxy A -3,2O-diketo steroid of the pregnane series which comprises mixing said steroid with an alkali metal borate to form a water-soluble steroid cycloborate, separating the said cycloborate, adding a mineral acid to produce a pH less than 2 and recovering the precipitated cis-16a,l7ot-dihydroxy steroid therefrom.

10. A process for the purification of an ILB-hydroxy cis-l6ot,17a-dihydroxy A -3,20-diketo steroid of the pregnane series which comprises mixing said Steroid with an aqueous alkaline borate solution to form a water-soluble steroid borate salt, separating the said borate salt in solution, acidifying to a pH less than 2, separating the resulting precipitate, dissolving the said precipitate in a lower alkyl alcohol, removing the lower alkyl alcohol borate formed and recovering the purified steroid therefrom.

11. The compound having the formula:

' onion onion CHzOH (13112011 I 0: 0:0 I I in which M is an alkali metal ion, which comprises treating 9ca-fiu0r0-l6a-hYdIOXY prednisolone with an aqueous alkali metal borate solution and separating said product therefrom. I

References Cited in the file of this patent UNITED STATES PATENTS 2,831,003 Thomas Apr. 15, 1958 

1. A PROCESS FOR THE SEPARATION AND PURIFICATION OF 9AFLUORO-16A-HYDROXYPREDNISOLONE WHICH COMPRISES ADDING SAID COMPOUND TO A SOLUTION CONTAINING BORIC ACID AND AN ALKALI METAL CARBONATE, FILTERING OFF ANY SOLIDS, ACIDIFYING TO A PH BELOW ABOUT TWO, REMOVING THE PRECIPITATE AND RECOVERING THE PURIFIED PRODUCT THEREFROM.
 11. THE COMPOUND HAVING THE FORMULA: 